Date of Award

12-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Physiology

Research Advisor

Gabor Tigyi, M.D., Ph.D.

Committee

Polly Hofmann, Ph.D. Anjaparavanda P. Naren, Ph.D. Abby L. Parrill, Ph.D. Christopher Waters, Ph.D.

Abstract

Lysophosphatidic acid (LPA) is a naturally occurring lipid mediator. It exists abundantly in biological fluids such as serum, saliva, follicular fluid, seminal fluid and malignant effusions and induces a vast array of biological responses affecting cell growth, survival, differentiation, migration and morphology. We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. Based on computational modeling octadecenyl thiophosphate (OTP) was synthesized: a novel rationally designed, metabolically stabilized LPA mimic. OTP was more efficacious than LPA in reducing g-irradiation-, camptothecin-, or TNF-a/cycloheximide-induced apoptosis and caspase 3, 8 and 9 activity in the IEC-6 cell line. The OTP- and LPA-elicited antiapoptotic effects were completely blocked by the MEK inhibitor PD98059 and the PI3K inhibitor LY294002. Pertussis toxin partially abolished OTP-induced ERK1/2 and apoptotic protection. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy g-irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase 3 positive cells but was ineffective in LPA2 knockouts. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but had no effect in LPA2 knockouts. Intraperitonealy administered OTP reduced death caused by LD100/30 radiation by 50%. Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype.

Unique sequence motifs in the LPA2 carboxyl-terminal (CT) enable it to form macromolecular complexes with PDZ and LIM domain proteins, which link it to G protein-independent signaling networks. Using deletion and site-specific mutagenesis, we mapped out C311xxC314 motif of LPA2-CT that is required for interaction with LIM domain proteins thyroid hormone receptor interactive protein 6 (TRIP6) and the proapoptotic molecule Siva-1 in vitro and in vivo. Palmitoylation that occurs on these cysteine residues, however, did not affect the association with TRIP6 or Siva-1. The L351A mutation in the PDZ motif weakened but did not abolish interaction with LIM proteins. Alanine mutation of the LIM binding motif or PDZ domain binding motif attenuated LPA-induced activation of the prosurvival ERK1/2 and Akt pathways in mouse embryonic fibroblasts (MEF) derived from LPA1 and LPA2 double knockout mice and reconstituted with mutants of LPA2. Neither of these mutations alone nor in combination had a detectable effect on G-protein-linked activation of Ca2+ mobilization. Triple alanine mutations modifying residues Cys311, Cys314, and Leu351 abolished the antiapoptotic effect of LPA. Together, these findings suggest the macromolecular complex formed between LPA2, TRIP6/Siva-1, and PDZ domain proteins plays an important role in mediating the anti-apoptotic effects of LPA2.

DOI

10.21007/etd.cghs.2008.0079

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